Several features of the nervous system specific 'growth-associated' protein GAP-43 suggest that it plays a fundamental role in cell biological processes common to axonogenesis and presynaptic function: It has been highly conserved during vertebrate evolution and is expressed in all neurons during development: Levels of GAP-43 are dramatically induced during axon outgrowth but, significantly, are not re-induced in injured axons of neurons that do not regenerate successfully: It is highly enriched in the growth cone where it associates with the membrane skeleton, the structure responsible for regulating the shape changes that are a crucial response to extracellular guidance cues: Finally, GAP-43 is a target for several Ca2+-dependent enzymes, including those responsive to extracellular signals, and levels of phosphorylated GAP-43 correlate with functional states of the growth cone. All of these features are consistent with a role for GAP-43 in the integration of extracellular signals within the growth cone, and suggest that understanding the molecular basis for GAP-43 function will further our understanding of the processes that direct axon growth. One powerful way to investigate GAP-43 function at the molecular level is to create a homozygous null mutant cell line in which transcription of GAP-43 is prevented, and then to compare its phenotype with cells that have further been transfected with GAP-43 cDNAs mutated at specific functional sites. Toward this end we have isolated and characterized a murine genomic clone for GAP-43 and used it to construct a replacement vector to knock out the GAP-43 gene at its native locus by homologous recombination. We will introduce the construct into the pluripotent embryonal carcinoma cell line P19 which differentiates into cholinergic-like neurons on treatment with retinoic acid, and will select lines homozygous fop disruption of GAP-43 transcription by their resistance to G418, a neomycin analog. Then, using a series of specific assays we have developed to investigate the role of GAP-43 in growth cone function, we will characterize the phenotype of the null mutant P19 cells with respect to attachment to laminin, regulation of neurite outgrowth and organization of the membrane skeleton and cytoskeleton. Finally we will transfect the P19 cells with GAP-43 cDNAs that have been mutated so that phosphorylation and calmodulin binding will be abnormal, and use these transfected cells to assess the relative contributions of phosphorylated GAP-43 and calmodulin to neurite outgrowth. The results generated by these experiments will provide information fundamentally important to our understanding of how axonogenesis is regulated during development and regeneration, and will enable us to begin to address the question of to what extent an axon needs to be structurally normal in order to form functional synapses.